Intraoperative, intravascular and endoscopic tumor and lesion detection, biopsy and therapy

ABSTRACT

Methods are provided for close-range intraoperative, endoscopic and intravascular detection and treatment of lesions, including tumors and non-malignant lesions. The methods use antibody fragments or subfragments labeled with isotopic and non-isotopic agents. Also provided are methods for detection and treatment of lesions with photodynamic agents and methods of treating lesions with a protein conjugated to an agent capable of being activated to emit Auger electron or other ionizing radiation. Compositions and kits useful in the above methods are also provided.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/293,313, filed on Aug. 22, 1994, now U.S. Pat. No. 5,716,595, whichis a continuation of U.S. Ser. No. 07/879,857, filed on May 6, 1992, nowabandoned. These cited applications are incorporated herein in theirentirety by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to improved methods for detectingand treating tumors and lesions and obtaining biopsy material in thecourse of intraoperative, laparoscopic, intravascular, and endoscopicexamination using a small detection instrument or monitor. In preferredembodiments, the methods utilize a labeled divalent single chainantibody fragment or subfragment with a molecular weight of 85,000daltons or less that specifically binds to an antigen produced by orassociated with the tumors or lesions. Alternatively, bispecific F(ab)₂and F(ab′)₂ fragments can also be used for pre-targeting according tothe present invention, if non-targeted fragments are cleared, and abivalent diagnostic hapten is then administered to facilitate targetdetection and possibly other procedures within 48 hours of the firstinjection.

[0004] 2. Description of the Prior Art

[0005] Surgical resection remains the primary curative approach in themanagement of cancer. Radioimmunodetection (RAID) is used to locate andstage tumors, and to monitor post-operative patients, by externalimaging, after injection of a radiolabeled antibody. Antibodies and/orantibody fragments which specifically bind antigens produced by orassociated with tumors (“anti-cancer antibodies”) are used as carriersfor radiolabels in RAID. It will be appreciated, that a tumor antigencan serve as a target for an antibody carrier even if it is not presentin serum in a detectable amount.

[0006] Resolution is affected by several factors that can limit the sizeof a tumor, especially a metastasis, which can be imaged by RAID.Non-invasive RAID is inherently limited by the distance between theradiation detector and the tumor. In the case of small, deep-seatedmetastatic tumors, this becomes the limiting factor in their detection.

[0007] Second-look surgery has been practiced where recurrence of apreviously excised primary tumor was indicated by elevated levels oftumor marker, e.g., carcinoembryonic antigen (CEA). Recently, a smallgamma detection probe has been developed which is capable of detectinggamma emission at short distances. Its intraoperative use in second-looksurgery has been reported to provide important information to thesurgeon for determining safe margins for resection and for detectingsmall metastases, by Aitken et al., Dis. Colon Rectum, 27,279-282(1984).

[0008] Nevertheless, elevated background radiation levels can interferewith and vitiate the advantage of short measuring distances in thistechnique. In addition, non-specific immunoglobulin uptake by tumortissue can complicate diagnosis.

[0009] U.S. Pat. No. 4,782,840 discloses a method for reducing theeffect of elevated background radiation levels during surgery. Themethod is to inject the patient with antibodies specific for neoplastictissue and which are labeled with radioisotopes having a suitably longhalf-life, such as Iodine-125. After injection of the radiolabledantibody, the surgery is delayed at least 7-10 days, preferably 14-21days, to allow any unbound radiolabeled antibody to be cleared to a lowbloodpool, background level.

[0010] U.S. Pat. No. 4,932,412 discloses methods for reducing orcorrecting for non-specific background radiation during intraoperativedetection. The methods include the administration to a patient who hasreceived a radiolabeled primary antibody, of a contrast agent,subtraction agent or second antibody which binds the primary antibody.

[0011] Tumors can be detected in body cavities by means of directly orindirectly viewing various structures to which light is delivered andthen collected. Lesions at any body site can be viewed so long asnonionizing radiation can be delivered and recaptured from thesestructures.

[0012] The prior art discloses improvements of such imaging approachesby using certain dyes that are accreted by lesions, such as tumors,which are in turn activated by a specific frequency of light. Theseimprovements are described in Dougherty et al., Cancer Res. 38:2628,1978; Dougherty, T. J., Photochem. Photobiol. 45:879, 1987; Jori andPerria, eds., Photodynamic Therapy of Tumors and Other Diseases; Padua:Libreria Progetto, 1985; Profio, Proc. Soc. Photoopt. Instr. Eng.907:150, 1988; Doiron and Gomer, eds., Porphyrin Localization andTreatment of Tumors; New York: Alan Liss, 1984; Hayata and Dougherty,Lasers and Hematoporphyrin Derivative in Cancer; Tokyo: Igaku-Shoin,1984; and van den Bergh, Chem. Britain 22:430, 1986, incorporated hereinin their entirety by reference.

[0013] These dyes are injected, for example, systemically, andlaser-induced fluorescence can be used by endoscopes to detect sites ofcancer which have accreted the light-activated dye. For example, thishas been applied to fluorescence bronchoscopic disclosure of early lungtumors (Doiron et al., Chest 76:32, 1979, included herein by reference;and references cited above).

[0014] It is known that dyes can be attached to antibodies for a morespecific binding to certain tissues and cells, including malignant andnormal cells, depending upon the discriminatory power of the antibodiesin question. In cancer, such labeled antibodies have been used in flowcytometry and in immunohistology to stain malignant cells with manydifferent kinds of anticancer antibodies, as described, for example, inGoding, Monoclonal Antibodies: Principles and Practice; London/New York,Academic Press, 1983; Ferrone and Dierich, eds., Handbook of MonoclonalAntibodies; Park Ridge, N.J., Noyes Publications, 1985; Wick and Siegal,eds., Monoclonal Antibodies in Diagnostic Immunohistochemistry; NewYork/Basel, Marcel Dekker, 1988, incorporated herein in their entiretyby reference. Fluorescent and other chromagens, or dyes, such asporphyrins sensitive to visible light, have been used to detect and eventreat lesions by directing the suitable light to the tumor or lesion(cited above). In therapy, this has been termed “photoradiation,phototherapy, or photodynamic therapy (Jori and Perria, eds.,Photodynamic Therapy of Tumors and Other Diseases, Padua: LibreriaProgetto, 1985; van den Bergh, Chem. Britain 22:430, 1986).

[0015] Monoclonal antibodies have been coupled with photoactivated dyesfor achieving a photodetection or photocopy (Mew et al., J. Immunol.130:1473, 1983; idem., Cancer Res. 45:4380, 1985; Oseroff et al., Proc.Natl. Acad. Sci. USA 83:8744, 1986; idem., Photochem. Photobiol. 46:83,1987; Hasan et al., Prog. Clin. Biol. Res. 288:471, 1989; Tatsuta etal., Lasers Surg. Med. 9:422, 1989; Pelegrin et al., Cancer 67:2529,1991 all incorporated in their entirety herein by reference). However,these earlier studies did not include use of endoscopic imaging and/ortherapy or biopsy applications, especially with the use of antibodyfragments or subfragments.

[0016] Further, there is a need in the art to utilize antibodies andantibody fragments that provide superior targeting specificity andaffinity but which are cleared quickly and naturally through the kidneysor which can be cleared quickly with clearing agents, so that targetingcan be effected within 48 hours.

[0017] A need continues to exist for simple methods which permitenhanced resolution to be achieved for close range intraoperative,intravascular, and endoscopic lesion detection, biopsy and therapy.

[0018] A need also exists for improved methods for detection and therapyof tumors.

[0019] A need also exists for methods to enable a clinician tointraoperatively, laparoscopically, intravascularly or endoscopicallydetect and treat non-malignant pathological lesions.

[0020] A need further exists for methods to accurately locate lesions ina patient to guide the biopsy implement to the lesion during a biopsyprocedure.

OBJECTS OF THE INVENTION

[0021] A principal object of the present invention is to provide methodsfor close-range intraoperative, laparoscopic intravascular, andendoscopic detection of tumors whereby discrimination between tumor andnon-tumor tissue is enhanced so that smaller tumors can be detected andappropriate margins can be determined more accurately to permitresection, irradiation, biopsy and/or tumor removal during the procedureand within hours of an injection of labeled protein, whereby surgical,intravascular, laparoscopic, endoscopic evaluation is not delayed.

[0022] Another object of the present invention is to provide a methodfor intraoperative, laparoscopic, intravascular or endoscopic detection,biopsy and therapy of tumors whereby the procedure can be selectivelyconducted within hours of the injection.

[0023] Another object of the present invention is to provide methods forclose-range intraoperative, laparoscopic, intravascular, or endoscopicdetection and treatment of non-malignant pathological lesions.

[0024] Another object of the invention is to provide improved lasertherapy, radioimmunotherapy, immunochemotherapy, and/or biopsyprocedures for lesions.

[0025] Upon further study of the specification and appended claims,further objects and advantages of this invention will become apparent tothose skilled in the art.

SUMMARY OF THE INVENTION

[0026] These objects can be attained in a method for close-range tumordetection, biopsy and treatment during an operative, laparoscopic,intravascular or endoscopic procedure. The method comprises injecting apatient subject to such a procedure parenterally with an effectiveamount of a labeled protein, such as an antibody fragment orsubfragment, which specifically binds a substance produced by orassociated with a targeted tumor. While the procedure is conducted, theaccessed interior of the patient is scanned at close-range with adetection means for detecting the presence of the labeled antibodyfragment or subfragment. The sites of accretion of the labeled antibodyfragment or subfragment are located by detecting elevated levels of thelabel at such sites with the detection means.

[0027] These objects can be attained in a method for close-rangeintraoperative, laparoscopic, intravascular, and endoscopic tumordetection, wherein a patient subject to such a procedure is injectedparenterally with an effective amount of a labeled antibody fragmentwhich specifically binds an antigen produced by or associated with atumor; the procedure is conducted, preferably, within 48 hours of theinjection when unbound radiolabeled antibody fragment in the patient'scirculation is reduced at least 50%; the surgically exposed,laparoscopically, or intravascularly or endoscopically accessed interiorof the patient is scanned at close range with a label detection means;and the sites of accretion of the labeled antibody fragments are locatedby detecting elevated levels of label at such sites with the detectionmeans.

[0028] In another related aspect, the invention provides a method forclose-range detection during an intravascular, operative, laparoscopic,or endoscopic procedure of a non-malignant pathological lesion, whereina patient subject to the procedure is injected parenterally with aneffective amount of an antibody fragment, subfragment or whole antibody,labeled with a labeling agent and which specifically binds an antigenproduced by or associated with the lesion, the accessed interior of thepatient is scanned at close-range with a detection means for detectingthe presence of the labeling agent, and the sites of accretion of thelabeled antibody fragments or labeled whole antibody are located bydetecting elevated levels of the labeling agent at such sites with thedetection means.

[0029] The invention also provides a kit suitable for use in anintraoperative, laparoscopic, intravascular, and endoscopic procedure.The kit comprises a first vial containing a sterile injectablepreparation for human use comprising an antibody fragment capable ofbeing conjugated with a labeling agent, and a second vial containing asterile injectable preparation for human use comprising a whole antibodycapable of being conjugated with a labeling agent different from thelabeling agent of the first vial.

[0030] In a related aspect, the invention provides a kit suitable foruse in an intraoperative, laparoscopic, intravascular, or endoscopicprocedure. The kit comprises a first vial containing a sterileinjectable preparation for human use comprising a labeled antibodyfragment and a pharmaceutically acceptable carrier, and a second vialcontaining a sterile injectable preparation for human use comprising awhole antibody labeled with a labeling agent and a pharmaceuticallyacceptable carrier, wherein the labeling agent of the second vialdiffers from the labeling agent of the first vial.

[0031] In another related aspect, the invention provides an improvedmethod of detection of lesions using endoscopic, laparoscopic,intravascular catheter or surgical procedures. The method comprisesinjecting a patient to undergo such a procedure with a protein, such asan antibody, antibody fragment or subfragment, labeled with afluorescent agent or dye, wherein the protein accretes at the lesion;permitting the labeled protein to accrete; and detecting the label witha light source supplied via the endoscope, laparoscope, or intravascularcatheter or during the surgical or during the biopsy procedure.

[0032] In another related aspect, the invention provides an improvedmethod of detection and treatment of lesions using an endoscope,laparoscopic, or intravascular catheter. The method comprises injectinga patient to undergo such a procedure with an agent capable of detectionwhich preferentially accretes at the lesion; permitting the agent toaccrete at the lesion; detecting the agent with a detection meanssupplied via the endoscope, laparoscopic, or intravascular catheter; andtreating the lesion by brachytherapy administered through the endoscope,laparoscopic, or catheter.

[0033] In another aspect, the invention provides an improved method oftreatment of lesions using endoscopic, laparoscopic, intravascularcatheter or surgical procedures. The method comprises injecting apatient to undergo such a procedure with an antibody or antibodyfragment labeled with a photoactive agent, wherein the antibody orfragment preferentially accretes at targeted lesions, permitting thelabeled antibody or fragment to accrete, and activating the photoactiveagent with a light source supplied via the endoscope, laparoscopic, orintravascular catheter or during the surgical procedure.

[0034] In another aspect, the invention provides an antibody fragmentcoupled with a photoactive agent.

[0035] In another aspect, the invention provides an improved method ofdetection and treatment of lesions using endoscopic, laparoscopic,intravascular catheter or surgical procedures.

[0036] The method comprises injecting a patient to undergo such aprocedure with a first composition comprising either a streptavidin- oravidin-conjugated antibody, biotinylated antibody to be used inconjunction with avidin and biotin, bifunctional antibody,antibody-hapten complexes, or enzyme-conjugated antibody, wherein theantibody is an antibody or antibody fragment which specifically binds amarker produced by or associated with the lesion; after the firstcomposition accretes at the targeted lesion, injecting a secondcomposition which bears a labeling agent capable of detection and whichcouples with the first composition; and detecting the label with adetection means via the endoscope, laparoscope, or intravascularcatheter or during the surgical procedure.

[0037] A variation of the above embodiment is when the first compositioncomprises biotinylated antibody or fragment and the second compositioncomprises biotin conjugated with fluorescent agent or dye. After thefirst agent accretes at the targeted lesion and prior to injecting thesecond composition, the patient is injected with a clearing compositioncomprising an agent to clear circulating biotinylated antibody orfragment.

[0038] In another aspect, the invention provides an improved method fordetection of lesions in a patient to undergo an endoscopic,laparoscopic, intravascular catheter or surgical procedure. The methodcomprises injecting a patient to undergo such a procedure with a firstcomposition comprising an antibody or fragment which preferentiallyaccretes at the lesion and which is labeled with an agent capable ofdetection with a photoscanning or magnetic resonance imaging device;imaging the lesion with the photoscanning or magnetic resonance deviceafter the labeled antibody or fragment accretes at the lesion; injectingthe patient with a second composition comprising an agent capable ofdetection by an endoscope, laparoscope, intravascular catheter ordetection device, such as a hand-held or portable detection means, andwhich preferentially accretes at the lesion, wherein the agent of thesecond composition is the same as or different from the agent of thefirst composition, and allowing the agent to accrete at the lesion; andusing the image of the lesion to determine the site for detecting theagent with a close-range detection means provided via the endoscope,laparoscope, or intravascular catheter or during the surgical procedure.

[0039] In another aspect, the invention provides an improved method fordetection of lesions in a patient to undergo an endoscopic,laparoscopic, intravascular catheter or surgical procedure. The methodcomprises injecting a patient parenterally with an antibody fragment orsubfragment specific to the lesion and which is labeled with a firstlabeling agent capable of detection using a detection device, and withan indifferent antibody fragment from the same or different species asthat used to prepare the specific antibody fragment, the indifferentfragment being labeled with a second labeling agent capable of beingindependently detected using a detection device, the labeling being soeffected that the kinetics and distribution of the labeled specificantibody fragment and the labeled indifferent antibody fragment in thepatient are substantially the same during the time period required fordetection, wherein at least one of the labeling agents is a photoactivedye; and during the procedure detecting for presence of the labelingagents in the patient with the detection device, the level of activityof the labeled indifferent antibody fragment being used to determine thedistribution of background activity due to non-targeted specificantibody fragment, whereby the activity of substantially only thetargeted lesion-localized specific antibody fragment is determined andsaid lesion is thereby detected and localized.

[0040] In another aspect, the invention provides an injectablecomposition, which comprises a substantially monospecific antibodyhaving a specific immunoreactivity of at least 70% to a marker producedby or associated with a lesion and a cross-reactivity tonon-lesion-associated antigens of less than 25%; the protein beinglabeled with a labeling agent capable of detection with a detectiondevice, said labeling being effected to an extent sufficient to reducethe specific immunoreactivity of the antibody by from 5 to 33%;indifferent protein from the same or different species as that used toprepare said specific antibody, said indifferent protein being labeledwith a labeling agent capable of independent detection by the detectiondevice; and a pharmaceutically acceptable injection vehicle wherein atleast one of the labeling agents is a photoactive dye.

[0041] In another aspect, the invention provides an improved method ofdetection and treatment of lesions. The method comprises injecting apatient with composition comprising a protein conjugated to an agentcapable of photoactivated to emit Auger electrons or other ionizingradiation, and, optimally, to an agent capable of detection, wherein theprotein conjugate accretes preferentially at the target lesion; andactivating the photoactivatable agent and detecting the optional agentcapable of detection.

[0042] In another aspect, the invention provides an improved method ofobtaining biopsy samples, comprising injecting a patient subjectparenterally with an effective amount of a labeled antibody fragment orsubfragment, which specifically binds an antigen produced by orassociated with a lesion, scanning the accessed interior of the patientat close range with a detection means for detecting the presence of thelabeled antibody fragment or subfragment, locating the sites ofaccretion of the labeled antibody fragment or subfragment by detectingelevated levels of the labeled antibody fragment or subfragment at suchsites with the detection means, and inserting a biopsy implement intoone or more located sites of elevated accretion to obtain a biopsysample.

[0043] In another aspect, the invention provides a method of detectionof lesions during an endoscopic, laparoscopic, intravascular catheter,or surgical procedure, comprising injecting a patient who is to undergosuch a procedure with a bispecific antibody F(ab)₂ or F(ab′)₂ fragment,wherein the bispecific antibody fragment has a first antibody bindingsite which specifically binds to an antigen produced or associated witha lesion, such as a tumor- or pathogen-associated antigen, and has asecond antibody binding site which specifically binds to a hapten,permitting the antibody fragment to accrete, optionally clearingnon-targeted antibody fragments using a galactosylated anti-idiotypeclearing agent if the bispecific fragment is not largely cleared fromcirculation within about 24 hours of injection, injecting hapten,preferably a bivalent hapten, wherein each hapten is a chelate orconjugate of a diagnostic radioisotope such as Tc-99m, I-123, In-11,Ga-67 or the like, or of a MRI image enhancing agent, e.g., Gadoliniumions, Lanthanum ions or the like, or other comparable label, whichquickly localizes at the target site and clears through the kidneys,detecting the presence of the hapten by detecting elevated levels ofaccreted label at the target sites with detection means within 48 hoursof the first injection, and conducting the procedure. BRIEF

DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 provides a representation of the expression vector, hMN-14dBpBEV.

[0045]FIG. 2 provides a representation of the expression vector,hMN-14(bs)hMu-9 dBpBEV.

DETAILED DISCUSSION

[0046] It must be recognized that embodiments of the present inventionrelate to intraoperative, laparoscopic, intravascular, and endoscopicexamination, biopsy and treatment of tissues and/or organs with alabeling agent detecting means capable of close approach to suspectedsites of tumor recurrence, metastasis or incomplete removal. As usedherein, endoscopic procedures are interpreted to include laparascopicprocedures.

[0047] Embodiments of the present invention also relate to theintravascular, intraoperative, laparoscopic, and endoscopic examinationof lesions with a labeling agent detecting means capable of closeapproach to suspected sites of the lesions, especially non-malignantpathological lesions. Lesions include cancer, infectious or inflammatorylesions, a non-tumorous or noninfectious inflammation, clots,hyperplasia and atherosclerotic plaques.

[0048] The methods of the present invention do not require processing ofimages, both target-specific and non-target-specific. Rather, oneembodiment enables a surgeon or clinician, through the use of, e.g., anintraoperative, laparoscopic, intravascular probe or an endoscope, toscan areas of suspected tumor growth relatively quickly and use thelevel of radiation to more precisely discriminate tumor tissue fromnon-tumor tissue and thereby more precisely define tumor borders forsurgical resection or diagnostic evaluation, or for laser or radiationtherapy, including brachytherapy and external beam therapy, or forimproved biopsy procedures.

[0049] Other embodiments enable the intravascular, intraoperative,laparoscopic, or endoscopic detection means to be similarly used todefine and treat lesions, especially non-malignant pathological lesions.

[0050] This general approach can be applied to all endoscopically,including laparoscopically, and even intravascularly approachablelesions or structures in the body Antibody fragments or subfragmentsthat specifically bind an antigen produced by or associated with alesion are useful in the present invention. Divalent single chainantibody fragments or subfragments with a molecular weight of 85,000daltons or less are preferred. The molecular weight can be 65,000daltons or less, 55,000 daltons or less or 50,000 daltons or less. Thefragment or subfragment can be monospecific or bispecific.

[0051] Additionally bispecific antibody F(ab)₂ or F(ab′)₂ fragments,wherein the bispecific antibody fragment has a first antibody bindingsite which specifically binds to an antigen produced or associated witha lesion, such as a tumor- or pathogen-associated antigen, and has asecond antibody binding site which specifically binds to a hapten, canbe used in the claimed procedures. If clearance is not sufficientlyrapid, e.g., within about 24 hours, a galactosylated anti-idiotypeclearing agent can be used to effect rapid clearance.

[0052] The use of protein-conjugates of light-sensitive dyes, especiallyof antibody fragments for imaging and fragments or intact antibodies,preferably of human or humanized forms, for optimization of early andsensitive tumor and other lesion detection or phototherapy in variousstructures of the body, particularly those reached by endoscopes orvarious intravascular or extravascular catheters, is preferred.

[0053] Further, in order to alleviate nonspecific targeting, which maybe experienced at times in some embodiments of the present invention,with such macromolecules as immunoglobulins, a method of controllingnonspecific antibody targeting, comprising the combination of a specificantibody and a nonspecific antibody, or fragments thereof, each labeledwith a different dye that is distinguishable from the other agent's dyeby photodetection methods, has now been developed. It is further shownthat the preferred localizing, specific antibody or fragment, can bedual-labeled with an isotope and a dye, either for simultaneous orseparate detection, as desired by the observer. The antibody orantibodies can be injected systemically with at least one dye attached,thus permitting different colors to be developed by photoactivation anviewed in the same endoscope by alternating the nature of the lightbeing delivered and collected for viewing. Such multipurpose viewingendoscodes have been described already, as for example in Hirano et al.,Lasers in the Life Sciences 3(2): 99, 1989, and in the van den Berghcitation above, both incorporated in their entirety herein by reference,but not in relation to antibody conjugates. A poster presented recentlyat the Seventh International Conference on Monoclonal AntibodyImmunoconjugates for Cancer, San Diego, Calif., Mar. 4-7, 1992, by Folliet al., claimed that a patient injected with a CEA monoclonal antibodyconjugated with fluorescein R showed green fluorescence of a rectaltumor observed by fluorescence rectosigmoidoscopy, and claimed thisdemonstrated the feasibility of photoimmunodiagnosis. Since such tissuesare known to have autofluorescence (as noted in the articles citedabove, e.g., van den Bergh, 1986), it is not verified that thisfluorescence is due to the fluorescing conjugate, and the use of wholeimmunoglobulin delayed imaging until background activity cleared.

[0054] In an embodiment of the present invention, more rapid endoscopicand laparoscopic imaging and detection are possible because of the useof antibody fragments and subfragments conjugated with suitablelight-activated dyes. This is also the case for light transmitted byintravascular catheters, both for detection and therapy of intravascularand perivascular lesions, either malignant or nonmalignant.

[0055] The use of whole immunoglobulins, even the smaller IgG forms,require sufficient time to clear from background, nonspecific organtargeting. This often constitutes a delay of considerable time (usually2 or more days) between injection of the chromogenic antibody andimaging of the localized dyes, which is not unlike the problemsexperienced in antibody imaging by external scintigraphy(radioimmunodetection).

[0056] It has now been discovered that the dyes and isotopes used forconjugation to proteins, especially antibodies, for lesion detection canbe more effective, faster, and safer when fragments or subfragment formsare deployed. Both monovalent and divalent antibody fragments andsubfragments are useful in the present invention. This rapid targetingthus permits the use of both short-lived and long-lived isotopes, ordyes of low and high stability after conjugation, since the detectionmethod can be employed within a few hours of the administration of thenew labeling agent(s).

[0057] Still another method to rapidly and selectively target adetecting agent by such ionizing and nonionizing radiation is by meansof antibody pretargeting procedures, such as, for example, streptavidin-or avidin-conjugated antibodies, biotinylated antibodies in conjunctionwith avidin and biotin, bifunctional antibodies, antibody-haptencomplexes, or enzyme-conjugated antibodies, reviewed in Paganelli, Nucl.Med. Commun. 12:211, 1991, incorporated herein in its entirety byreference.

[0058] In addition to delivering radiation to tumors by such 2- and3-step procedures, the methods can be used to achieve hightarget/nontarget ratios for intraoperative, endoscopic, andintravascular detection and therapy of tumors and other lesions,including the use of photoradiation for tumor and lesion detection andtherapy by endoscopic and intravascular probes within scopes andcatheters. The use of light and porphyrins in cancer therapy has beenreviewed by the references already cited above.

[0059] When endoscopes are used to deliver the nonionizing radiation fortherapy of the dye-containing tumors, or for the detection of tumors byfluorescence endoscopy, the procedures are limited to lesions that areaccessible to the exciting light and to the detection of the emittedfluorescence, such as in the oral cavity, trachea, bronchi, esophagus,colon, rectum, bladder, vagina, uterus, and other body cavities by useof laparoscopes. Use of light-bearing catheters inserted through veinsor arteries enables a more extensive application, especially into organsthat are currently examined by intraarterial radiological procedures.Therapeutic applications of antibodies conjugated with light-activatingdyes can also be achieved by delivering the nonionizing radiation viasuch intravascular catheters, particularly in cases where there is apathological blockage, such as in atherosclerotic plaques orintravascular thrombi or emboli.

[0060] This invention also includes the therapeutic use of proteins,especially a bifunctional antibody or antibody fragment, conjugated toagents which can be photoactivated to emit Auger electrons or otherionizing radiation, and, optimally, to agents capable of detection.After the conjugate accretes at the targeted site, activating means andan optional detection means are provided. Agents capable ofphotoactivation include halogenated compounds, such as halogenatedpyrimidines, and certain stable elements, such as iodine and indium. Theactivating means include monochromatic x-rays, especially those emittedin an energy range of 20-70 keV, more preferably 30-40 keV. The 2- and3-step procedures disclosed above can be used to deliver agents whichcan be photoactivated to the targeted sites. These embodiments may applyto intraoperative and endoscopic as well as more generalized uses.

[0061] Another goal of an embodiment of the invention is theachievement, as early as possible, of a high target/nontarget ratio ofthe dye, so that the photoradiation procedure can be instituted beforethe loss of the dye in the tumor due to instability of the conjugate orbecause of high autofluorescence in surrounding normal tissues. By usingan intravascular catheter, one can now deliver nonionizing radiation tomost structures in the body which are accessible to an intravascularcatheter. In terms of a rapid method of targeting the dye to a tumor orlesion, it is now discovered that the preferred method is the use ofantibody fragments and subfragments as the delivery vehicle of the dye(as a chromophore conjugate) or to use a pretargeting system, such as a3-step biotin-avidin procedure. In the latter, for example, thebiotinylated antitumor antibody is injected parenterally. Aftersufficient time for the reagent to localize to tumor optimally, such asafter about 24 hours and up to about 7 days, but more preferably after48 hours and before 96 hours, a dose of cold (unlabeled) avidin is givenparenterally, followed at 24-96 hours later by a biotin derivativelabeled with the detection or therapy isotope or with a suitablephotoirradiation dye. The radioactive biotin can be given for detectionof lesions intraoperatively, laparoscopically, or endoscopically, orbiotin conjugated with a chromophore can be given for tumor/lesiondetection and/or therapy by a laparoscope, an endoscope or anintravascular catheter. A description of the 3-step system for targetingcarcinoembryonic antigen in tumors, using biotin labeled with In-ill,has been described by Paganelli et al., Cancer Res. 51:5960, 1991,incorporated herein by reference. Such pretargeting methods have notbeen described previously for improved endoscopic and/or intravasculardetection and/or therapy of cancer and other pathological lesions.Bispecific pretargeting methods are also contemplated, such as thosedisclosed in U.S. Pat. Nos. 5,256,395 and 5,274,076, and Rouvier et al.,Hormone Research, 47:163167, 1997, incorporated herein in their entiretyby reference, but with the added improvement of rapid clearance,optionally with a galactosylated anti-idiotype clearing agent, therebypermitting injection of an imaging hapten and localization of the lesionwithin 48 hours. A suitable galactosylated anti-idiotypic antibody isdescribed in U.S. application Ser. No. 08/486,166 and U.S. Pat. No.4,859,449, both of which are incorporated herein by reference.

[0062] It has also been discovered that more effective detection andtherapy can be achieved with combinations of agents and proceduresinvolving photodynamic detection and therapy, with or without the use ofantibody conjugates, and radioimmunotherapy and/or chemoimmunotherapy.Photodynamic therapy has been used in combination with radiation therapyand chemotherapy in cancer patients (Hayata and Dougherty, 1984, citedabove), but combining photodynamic therapy with radioimmunotherapyand/or chemoimmunotherapy is even more effective, since the isotopes ordrugs conjugated to tumor-seeking antibodies are more specificallyeffective than external-beam irradiation or free-drug chemotherapy. Themethods and agents for radioimmunotherapy and for chemoimmunotherapy aredescribed in patents and applications, such as, U.S. Pat. No. 4,331,647,U.S. Pat. No. 4,818,709, U.S. Pat. No. 4,348,376, U.S. Pat. No.4,361,544, U.S. Pat. No. 4,444,744, U.S. application Ser. No.07/182,623, referred to as the “Goldenberg” patents and “Hansen”application, incorporated herein by reference.

[0063] An improved method of cancer therapy is achieved when thephotodynamic therapy involves photosensitive agents conjugated totumor-seeking antibodies combined with radioimmunotherapy and/orchemoimmunotherapy, since these all entail more specific cancer therapymodalities that involve higher therapeutic indices as compared toconventional therapeutic modalities.

[0064] General methods of labeling antibodies with fluorochromes areknown in the art, and can be found, for example, in Weir, ed., Handbookof Experimental Immunology, Vol. 1, Chapter 28, pp. 28.1-28.21, Oxford,Blackwell Scientific, 1986, incorporated herein in its entirety byreference. For therapeutic purposes, the delivery of high numbers ofphotoactivating agents to the lesions (malignant and nonmalignant) iscritical. Increasing the number of photoactivating molecules attached tothe immunoglobulin can affect the antibody's immunoreactivity and, inturn, targeting properties, and usually requires that fewer than 10 suchmolecules be conjugated to the immunoglobulin. Another requirement is toattach the photoactivating molecules away from the antigen-bindingregion of the antibody, so as not to hinder the antibody's bindingregion of the antibody, so as not to hinder the antibody's binding andtargeting properties. Both of these requirements are met by use of theaminodextran conjugation method described by Shih et al. in U.S. Pat.No. 5,057,313, incorporated herein by reference. This method permitshigh numbers of fluorochromes and other light-activating agents to beconjugated to the carbohydrate region of the antibody, away from theantigen-binding sites, usually more than 20 molecules per IgG molecules,and sometimes considerably more.

[0065] Previously, Oseroff et al. (Proc. Natl. Acad. Sci. USA 83:8744,1986) used a photosensitizer, chlorine, conjugated in high numbers toantibody via a dextran linkage, and showed photodestruction of humanT-cell leukemia cells in vitro. In this circumstance, light could bedelivered to the leukemic cells in culture. However, leukemic cells inhumans are not accessible to such phototherapy in their usual reservoirsin the body, so it is not readily apparent how these leukemic cellculture experiments would be extrapolated to the human situation.Indeed, in vivo experiments in animals have shown that photodynamictherapy affects the vascularization of tumors by reducing blood flow(Doiron and Gomer, eds., Prog. Clin. Biol. Res., p. 170; New York, A. R.Liss, 1984). Indeed, van den Bergh (cited above) has cautioned “possibleextrapolation of PDT (photodynamic therapy) results from in vitroexperiments to the situation in vivo.” Similarly, it is difficult toextrapolate from animal experiments to the clinic, since it is wellknown that rodent models do not bear the same distribution of targettumor-associated antigens in their blood and tissues as is true forhumans, and that the tumor uptake of antibody conjugates is hundreds tothousands and more higher than in humans.

[0066] The procedures of intraoperative detection involving the methodsand reagents described herein can be combined optimally with externalimaging by radioimmunodetection (RAID), using preferably ^(99m)Tc-,¹¹¹In-, or ¹²³I-labeled antibody fragments or subfragments, andperforming a scanning of the patient prior to surgery, preferably within24 hours of surgery. The RAID study provides the surgeon withinformation regarding sites of abnormal radioactivity of putativedisease, which enables the surgeon to focus in on such areas with theintraoperative probe during surgery or laparoscopy. Likewise, the RAIDstudy can be advantageously performed prior to the fluorescenceendoscopy or intravascular catheterization study, in order to betterdefine areas of disease. More versatile agents can also be used, wherebyan anticancer antibody fragment is labeled with both an imaging anddetection isotope (e.g., ^(99m)Tc) and with a fluorescent dye, or wherea bifunctional or bispecific antibody is used, one arm directed to thelesion and labeled with a suitable radionuclide, and the other armdirected to a hapten conjugated with a fluorescent dye, or to a haptento which a fluorescent dye can be bound after injection parenterally. Inanother combination, one arm of a bispecific F(ab)₂ or F(ab′)₂ antibodyfragment is directed to the lesion, e.g., a tumor, and the other arm toa hapten, the hapten to be administered in a susbsequent step, afterrapid clearance. Other combinations of an isotopic probe and afluorescent (or chromophore) probe, as apparent from 2- and 3-steptargeting methods (cited above), can be used to allow forcontemporaneous use of nonionizing and ionizing radiation probes forintraoperative, endoscopic, laparoscopic, and intravascular detection ofpathological lesions.

[0067] This principle is also applicable to therapy, where combinationsof modalities are more effective than one method alone. For example,systemic radioimmunotherapy or chemoimmunotherapy can be advantageouslygiven before or after photoactivated therapy (via an endoscope,laparoscope, or intravascular catheter after deposition of thechromophore by conventional injection or via an antibody conjugate),whereby the radioimmunotherapy or chemoimmunotherapy are working incombination or synergistically with the phototherapy. These variationsagain can include 2- and 3-step pretargeting procedures, includingbispecific antibodies, in order to achieve higher target/nontargetratios for the delivery of the chromophore or of the radiation to thetumor or pathological lesion.

[0068] Still another improvement in cancer therapy is the use ofendoscopes, including laparoscopes, and catheters involvingisotopically- or photosensitizer-conjugated anticancer antibodies incombination with brachytherapy involving the implantation of radioactivepellets by means of a scope or catheter.

[0069] The isotopically or photosensitizer-conjugated antibody can serveas a detection method for directing the application of brachytherapy, oralso as a therapy in conjunction with brachytherapy.

[0070] Such brachytherapy is conducted in situations almost identical tothe use of photodynamic therapy, so it is an improvement to usephotodynamic therapy, with or without conjugating the photosensitizer toa tumor-seeking antibody (but preferably with an antibody conjugate),together with brachytherapy, such as radon-222, iodine-125, cesium-137,cobalt-60, and iridium-192.

[0071] It is preferred to use brachytherapy in combination with lasertherapy, external beam radiation, chemoimmunotherapy and/orradioimmunotherapy, since higher antitumor doses with lower host sideeffects are achieved. Combinations of brachytherapy with Nd-YAG lasertherapy in lung carcinoma have already been reported (Allen et al., Am.J. Surg. 150:71, 1985; Schray et al., Int. J. Radiat. Oncol. Biol. Phys.11:403, 1985 both incorporated in their entirety herein by reference),as well as the general use of intraluminal brachytherapy in lung cancerby Nori et al., Surg. Clin. N. Amer. 67:1093, 1987, incorporated hereinin its entirety by reference. More effective therapy is now achieved bycombinations of the various modalities of brachytherapy, laser therapy,photodynamic therapy, radioimmunotherapy, and chemoimmunotherapy,particularly with the application of tumor-seeking antibodies conjugatedwith therapeutic modalities.

[0072] The short-range detection method according to the invention mayuse a detector capable of detecting radiation emitted from or colorprovided by a label bound to the protein.

[0073] The label may be alpha, gamma, beta, positron, fluorescence orany other detectable ionizing or nonionizing radiation which can beproduced by a convenient label capable of attachment to a protein,preferably an antibody or antibody fragment. For example, a gammadetector suitable for such a function has been reported by Aitken etal., Dis Colon & Rectum, 27, 279-282(1984). These authors describe ahand-held gamma probe using a cadmium telluride scintillation crystal, apreamplifier and an amplifier with a digital readout displaying theradioactive counts (Radiation Monitoring Devices, Watertown, Mass.). Thescintillation crystal is housed in a 16-mm diameter lead collimator witha 4-mm aperture. This device has been shown to detect tumor withradiolabeled antibody injected intraperitoneally, and to distinguishtumor tissue from nearby non-tumor tissue. However, the disclosed methodrequires long delays between injection and probing, with unpredictableresults obtained from patient to patient.

[0074] It will be apparent to the ordinary skilled artisan that otherdetection means can be used and that the detector is not limited togamma radiation.

[0075] It will also be apparent that the detector can be made todiscriminate between radiolabeling agents of different energies ofincident radiation, e.g., between gamma radiation in different rangeswithin the broad 201,000 keV range which is used for gamma scintillationcounters and/or between alpha-, gamma-, and beta-radiation emitted bydifferent radiolabeled proteins. Thus, the invention is not limited bythe type of detector used.

[0076] A scintillation crystal can be mounted on the end of a fiberoptic cable and its optical response to incident gamma radiation can betransmitted to a photomultiplier and associated circuitry through theoptical fiber. This can reduce the size of the detector to be compatiblewith use in conjunction with an endoscope or intravascular catheter. Theendoscope or catheter can be shielded to serve as a collimator, wherenecessary, and/or fitted with a window at a known distance from itsterminus, with the scintillation crystal housed therein.

[0077] Various other modifications and adaptations of the foregoing willbe readily apparent to the ordinary skilled artisan, in light of theparticular needs of the situation. However, the method of the presentinvention is not limited by any specific type of radiolabeling agentdetector. Rather, any detector which is capable of detecting the labellocalized in targeted lesion, tissue or organ can be used.

[0078] The detection means can be used in the form of an endoscope, andinserted into a body cavity through an orifice, such as, the mouth,nose, ear, anus, vagina or incision. The term “endoscope” is herein usedgenerically to refer to any scope introduced into a body cavity, e.g.,an anally introduced endoscope, an orally introduced bronchoscope, aurethrally introduced cystoscope, an abdominally introduced laparoscopeor the like. Certain of these may benefit greatly from further progressin miniaturization of components and their utility to practice themethod of the present invention will be enhanced as a function of thedevelopment of suitably microminiaturized components for this type ofinstrumentation.

[0079] Highly miniaturized probes which could be introducedintravascularly, e.g., via catheters or the like, are also suitable foruse in the embodiments of the invention for localizing and treatingtumors and the embodiments for localizing and treating non-malignantpathological lesions, such as, an infarct, including myocardial,atherosclerotic plaque, clot, including thrombosis, pulmonary embolism,infectious or inflammatory lesion, non-tumorous or noninfectiousinflammation, or hyperplasia.

[0080] In methods of the present invention, techniques useful in imagingapplications are adapted for the improved methods of the presentation.Methods of localization and therapy of tumors and lesions, and methodsof organ imaging, using radiolabeled antibodies and antibody fragmentswhich specifically bind markers produced or associated with tumors,lesions and normal organs or tissue, have been disclosed, inter alia, inU.S. application Ser. No. 07/694,977, filed May 6, 1991 and U.S. Pat.No. 4,331,647; 4,348,376; 4,361,544; 4,444,744; 4,460,561; and4,624,846, the disclosures of all of which are incorporated herein theirentireties by reference (hereinafter, “the Goldenberg patents”). Thesereferences also disclose antibodies and antibody fragments for use inthe foregoing methods, together with methods for obtaining them and forradiolabeling them with appropriate radionuclides.

[0081] Antibody fragments useful in the present invention are F(ab′)₂F(ab)₂, Fab′, Fab, Fv and the like, including hybrid fragments. Alsouseful are any subfragments retaining the hypervariable, antigen-bindingregion of an immunoglobulin and having a size similar to or smaller thana Fab′ fragment. This will include genetically engineered and/orrecombinant proteins, whether, single-chain or multiple-chain, whichincorporate an antigen binding site and otherwise function in vivo astargeting vehicles in substantially the same way as naturalimmunoglobulin fragments. Such single-chain binding molecules aredisclosed in U.S. Pat. No. 4,946,778, which is hereby incorporated byreference. Fab′ antibody fragments may be conveniently made by reductivecleavage of F(ab′)₂ fragments, which themselves may be made by pepsindigestion of intact immunoglobulin. Fab antibody fragments may be madeby papain digestion of intact immunoglobulin, under reducing conditions,or by cleavage of F(ab)₂ fragments which result from careful papaindigestion of whole Ig. The fragments may also be produced by geneticengineering.

[0082] An Fv fragment is approximately 25,000 daltons and is thesmallest fragment produced from IgG and IgM that contains a completeantigen binding site. Fv fragments have the same binding properties andsimilar three-dimensional binding characteristics as Fab (50,000daltops). The V_(H) and V_(L) chains of the Fv fragments are heldtogether by noncovalent interactions. These chains tend to dissociateupon dilution, so methods have been developed to crosslink the chainsthrough glutaraldehyde, intermolecular disulfides or a peptide linker.However, the divalent single chain antibodies of the present inventionare produced by recombinant techniques.

[0083] Divalent single chain antibody fragments or subfragments, such as(sFv)₂ and (sFv′)₂, are preferred antibodies to utilize in the methodsof the present invention. The (sFv)₂ and (sFv′)₂ antibody fragments havetwo antigen binding sites and provide better targeting and affinity tothe antigen than monovalent antibodies with one antigen binding site,such as Fab′, Fab, Fv or Fv′. The (sFv)₂ and (sFv′)₂ antibody fragmentsare preferred because they have a lower molecular weight than F(ab′)₂fragments (IgG—110,000 daltons) resulting in faster clearance from thebody. The fragments, (sFv)₂ and (sFv′)₂, are similar in molecular weightto the Fab′ (55,000 daltons) but are divalent rather than monovalent,thus providing an antibody fragment with better affinity and specificityto the lesion-associated antigen. An (sFv′)₂ fragment has a cysteine atthe end of each Fv (Fv′). Each is independently expressed recombinantly,e.g., in E. coli, and two Fv′ fragments are chemically joined through adisulfide bond. See, e.g., Tai et al., Cancer Research (Suppl.)55:5983s-5989s, 1995; and Zhu et al., Biotechnology, 14:192-196, 1996,both of which are incorporated herein by reference.

[0084] A F(ab)₂ or F(ab′)₂ fragment is too large to be filtered throughthe glomerular basal membrane. This divalent fragment must becatabolized elsewhere; e.g. in the liver, and the smaller breakdownproducts are excreted via the kidneys. Without the use of a clearingagent, the clearance of this fragment takes longer than the divalentsingle chain fragments of the present invention and can unduly delayother procedures.

[0085] The divalent single chain antibody fragments or subfragmentsuseful in the present invention have a molecular weight of 85,000daltons or less, which is about the upper limit for filtration by thekidneys. The molecular weight of these antibody fragments can be 65,000daltons or less, 55,000 daltons or less or 50,000 daltons or less. Thefragment or subfragment can be monospecific meaning that the two antigenbinding sites are directed to epitopes on one antigen, such as thefollowing antibodies, MN14, LL2, MA5, and RS7 or they can be bispecificwhere each antigen binding site binds to an epitope on a differentantigen, such as hMN14/hMu9 (h is an abbreviation for human). Themethods of making and the description of these antibodies are disclosedin U.S. Ser. Nos. 08/690,102 and 08/318,157, which are both incorporatedherein in their entirety by reference.

[0086] However, bispecific F(ab)₂ and F(ab′)₂ divalent fragments alsocan be used, wherein one binding site is specific to a tumor or lesionand the second binding site is specific to a hapten; e.g., a diagnosticagent, which is radiolabeled, fluorescent labeled, or Auger generatinglabeled hapten. In order to effect location of the target site within 48hours, the non-targeted bivalent F(ab)₂ and F(ab′)₂ fragments, if notsignificantly cleared (at least 50% reduction in circulating level,preferably 85% or more) within about 24 hours, are cleared fromcirculation using any one of several known clearing agents. In apreferred embodiment, an anti-idiotype antibody is used, preferably onewhich specifically binds the target binding site of the bispecificfragment. The anti-iodiotype antibody is preferably modified byconjugation to galactosyl residues for more rapid uptake in the liver.Alternatively, this bispecific antibody can be biotinylated andclearance can be effected with avidin or streptavidin. A furtheralternative is the use of a second antibody clearance. For specifics onthis type of clearance methods, see U.S. Pat. No. 4,624,846, Goldenberg,which is herein incorporated by reference in its entirety.

[0087] The following procedures provide examples of the construction ofsingle chain Fv homodimeric and heterodimeric diabodies.

[0088] Single-Chain Antibody (scFv) for hMN-14:

[0089] In the original design for hMN-14 scFv, the VK domain is linkedat its C-terminus to the VH by a 15-mer peptide linker having thesequence (GGSGS)₃. The DNA sequence encoding the hMN-14 scFv is clonedinto a pET-related bacterial expression vector (Novagen, Madison Wis.),in which the gene encoding hMN-14 scFv is driven by a T7 promoter. ThescFv is obtained by solubilization and refolding of inclusion bodyproteins from the host E. coli BL21 (λDL3)(Studier and Moffatt, J. Mol.Biol. 189:113, 1986) as described (Buchner et al., 1992). Properlyrefolded proteins are purified by sequential ion exchange chromatographyon Mono Q (Pharmacia) followed by size exclusion chromatography on a TSKG3000SW (TosoHaas) column. The scFv is purified as a monomeric proteinwith immunoreactivity comparable to that of hMN-14 Fab′ fragment.

[0090] Design and Construction of Bacterial Express Vector forHomodimeric Diabody for hMN-14:

[0091] A pUC19-based vector, hMN-14pBEV (Leung et al., Cancer Res.(suppl.) 55:5968s-5927s, 1995), containing the sequence encoding the Fabfragment of hMN-14, is used as the template for the PCR-amplification ofthe hMN-14 VK and VH sequence. The PCR-amplified VK sequence is linkedat its C-terminus to the VH sequence via a five amino acid linker,GGPGS. The resultant protein, if expressed, should have a configurationof VK-linker-VH. Other linkers, such as those derived from theN-terminus of the human CK domain (RTVAA, RTVAAPS or RTVAAPSHFT) canalso be used. This shortened linkers are designed to prevent intra-chainand to encourage inter-chain cross-over pairing of VK and VH.Alternatively, the diabody can be aligned in the configuration ofVH-linker-VK.

[0092] To construct the diabody expression vector, the DNA sequenceencoding the Fab for hMN-14 is spliced out from the bacterial expressionvector hMN-14pBEV by restriction digestion, and is replaced by thatencoding the diabody. A PelB sequence is joined in-frame to theN-terminus of the diabody gene to allow transportation of the translatedproduct into the bacterial periplasm for proper folding and disulfide-linkage formation (Better et al., Science 240:1041-1043, 1988;Skerra & Plückthun, Science 240:1038-1041, 1988). The diabody gene isdriven by an inducible LacZ promoter. The structure of the finalexpression vector, hMN-14 dBpBEV, is shown in FIG. 1.

[0093] Design and Construction of a Bacterial Expression Vector for aBispecfic Diabody for hMN-14 and hMu-9:

[0094] The V-region sequences for a colon-specific antigen-p antibody,Mu-9, has been elucidated (Krishnan et al., Cancer (suppl.)80:2667-2674, 1997). A humanized Mu-9 (hMu-9) antibody was derived andshown to retain full immunoreactivity as compared to that of murineMu-9. The expression vector for hMu-9, designated as hMu-9 KpdHL2, isused as the template for the PCR-amplification of its V-gene sequences.Unlike the homodimeric expression vector for hMN-14 diabody, theexpression vector for the bispecific diabody contains a dicistronic genesequences with the configuration: VL(hMN-14)-linker-VH(hMu-9), alignedin series with VL(hMu-9)-linker-VH(hMN-14) (FIG. 2). The linker iscomposed of the sequence GGPGS. The shortened linker is designed toprevent intra-chain pairing of VK and VH and to ensure cross-overinter-chain pairing. Both gene sequences are fused in-frame with a PelBsequence at their N-terminus. The dicistronic gene is cloned into thehMN-14pBEV vector, replacing the sequence encoding the Fab fragment. Thefinal expression vector is designated as hMN-14(bs)hMu-9 dBpBEV (FIG.2).

[0095] Expression and Purification of Monospecific and BispecificDiabodies:

[0096] CsCl-purified DNAs of hMN-14 dBpBEV and hMN-14(bs)hMu-9 dBpBEVbacterial expression vectors are used to transform the E. coliBMH71-18mutS (CLONTECH, Palo Alto, Calif.). Transformants are grownovernight at 37° C. in 2×TY supplemented with 55 mM glucose and 100μg/ml of ampicillin. The overnight culture is pelleted, washed twicewith 2×TY, and resuspended with prewarmed 2×TY in the presence of 1-5 mMisopropylthiogalactoside. After an induction period of 20-24 h at 37°C., the culture is pelleted. The diabody in the periplasm is releasedfrom the cell pastes by osmotic shock facilitated by partial digestionof the cell wall with chicken egg white lysozyme (Carter et al.,Bio/Technology 10: 163-167, 1992). The diabody is then affinity purifiedby Staphylococcal protein A chromatography. For the bispecific diabody,the protein A-purified proteins should contain a mixture of bispecificdiabody and each of the inactive homodimers. The mixture is subjected toanother round of affinity-purification with WI2-column. WI2 is ananti-idiotype antibody specific for MN-14 (Losman et al., 1994). Sincenone of the two possible homodimers is immunoreactive, allimmunoreactive diabodies have to be bispecific.

[0097] An additional example of divalent single chain antibody fragmentsuseful in the methods of the present invention are disclosed in Adams etal., Cancer Research 53:4026-4034 (1993) and incorporated herein byreference. This publication discloses preparing divalentdisulfide-bonded (sFv′)₂ molecules which exhibit divalent bindingcharacteristics and equivalent binding affinity to parental IgG. Thesemolecules have a molecular weight of 53,000 daltons and remain below thethreshold for renal clearance. They are cleared from the blood nearly asrapidly as sFv monomers, apparently without in vivo disruption of the(sFv′)₂ interchain disulfide bond.

[0098] Another useful single chain divalent antibody fragment isdisclosed by Haunschild et al., Antibody, Immunoconjugates, andRadiopharmaceutics, Vol. 9, No. 2, 111-128 (1995) and incorporatedherein by reference. These antibody fragments are bivalent“miniantibodies” based on single chain Fv fragments that are fused via aflexible hinge-region to association domains. This antibody is known asscZIP and is held together by the formation of a parallel coiled coilhelix, also termed leucine zipper. These antibodies were found to becleared through the kidneys somewhat slower than a scFv fragment butmuch faster than a whole antibody.

[0099] A further single chain Fv useful in the presently describedmethods is described in Immunology/Preclinical and Clinical BiologicalTherapy 1, Abstract #180 by Rajagopal et al., incorporated herein byreference. The disulfide-stabilized (dsFv) fragments of antibodies aremore stable than their single-chain counterparts. The heavy and lightchains are held together by the conventional peptide linker andcysteines are engineered into the heavy and light chain variable regionsso that a disulfide bond is formed between the two chains.

[0100] Bispecific divalent single chain antibodies (diabodies) alsouseful in the present invention are disclosed in Tai et al.l, CancerResearch (suppl.) 155:5983s-5989s (1995). The molecular size of thesediabodies are less than Fab′ when evaluated by molecular sizing gelexclusion (FIG. 3 of the Tai et al. publication).

[0101] The antibody may be whole immunoglobulin of any class, e.g., IgG,IgM, IgA, IgD, IgE, chimeric or hybrid antibodies with dual or multipleantigen or epitope specificities. It can be a polyclonal antibody,preferably an affinity-purified antibody from a human or an appropriateanimal, e.g., a primate, goat, rabbit, mouse or the like. Monoclonalantibodies are also suitable for use in the present method, and arepreferred because of their high specificities. They are readily preparedby what are now considered conventional procedures of immunization ofmammals with immunogenic antigen preparation, fusion of immune lymph orspleen cells with an immortal myeloma cell line, and isolation ofspecific hybridoma clones. More unconventional methods of preparingmonoclonals antibodies are not excluded, such as interspecies fusionsand genetic engineering manipulations of hypervariable regions, since itis primarily the antigen specificity of the antibodies that affectstheir utility in the present invention. It will be appreciated thatnewer techniques for production of monoclonals can also be used, e.g.,human monoclonals, interspecies monoclonals, chimeric (e.g.,human/mouse) monoclonals, genetically engineered antibodies and thelike.

[0102] It should be noted that mixtures of antibodies, isotopes, andimmunoglobulin classes can be used, as can hybrid antibodies. Thehybrids can have two different antigen specificities, e.g., one armbinding to a tumor antigen such as carcinoembryonic antigen and anotherarm binding to another antigen, e.g., CSAP, or one arm could bind to oneepitope on, e.g., carcinoembryonic antigen and the other arm could bindto another carcinoembryonic antigen epitope. The foregoing are merelyillustrative, and other combinations of specif icities can be envisionedthat also fall within the scope of the invention.

[0103] Hybrid antibody fragments with dual specificities can be preparedanalogously to the anti-tumor marker hybrids disclosed in U.S. Pat. No.4,361,544. Other techniques for preparing hybrid antibodies aredisclosed in, e.g., U.S. Pat. No. 4,474,893 and 4,479,895, and inMilstein et al., Immunol. Today, 5,299(1984).

[0104] Unbound labeled antibody fragments will clear from the patient'scirculation quickly and thereby be reduced by at least 50% within 48hours, preferably 24 hours, more preferably 12 hours, and even morepreferably 6 hours, after injection. It is preferable that the reductionbe at least 75%, more preferable 85%.

[0105] Unbound whole antibody will clear from the patient's circulationand the amount of unbound whole antibody reduced by no more than 50%, inno less than 48 hours.

[0106] The labeling agents useful in the methods of the presentinvention include isotopic or non-isotopic labels, such as fluorescentagents and other dyes, etc. Such labels are well known to these skilledin the art.

[0107] The radioisotope selected by one skilled in the art to radiolabelthe fragment will have a half-life to complement binding time of thefragment to the antigen.

[0108] The labeling agent for the antibody fragment is preferably anisotope with a gamma radiation emission peak in the range of 20-500 keV,primarily because the state of the art for radiation detectors currentlyfavors such radiolabels.

[0109] Suitable such radioisotopes for antibody fragments include, e.g.,Iodine-123, Indium-113m, Gallium-68, Rheniua-188, Technetium-99m, andFluorine-18. Preferred isotopes are Technetium-99m and Iodine-123.However, isotopes with longer half-lives can also be used. Beta emittersand position emitters also can be used.

[0110] The labeling agents used in the methods of the present inventioninclude isotopic or non-isotopic labels, such as fluorescence, etc. Suchlabels are well known to these skilled in the art and are disclosedabove.

[0111] The radioisotope selected by one skilled in the art to radiolabelthe whole antibody should have a half-life which complements theantibody being used.

[0112] Examples of suitable radioisotopes for whole antibodies includeIndium-111, Iodine-125, Iodine-131, and Gallium-67.

[0113] In a method of the present invention wherein more than oneisotope is used, the two radiolabels should be of sufficiently differentenergies or sufficiently different half-lives to be separatelydetectable with the detection means. Suitable such pairs ofradioisotopes include, e.g., Iodine-131/Iodine-123,Iodine125/Technetium-99m and the like.

[0114] U.S. Pat. No. 4,782,840 discloses antibodies specific forneoplastic tissue and radioisotopes useful in a surgical procedurewherein an animal suspected of containing neoplastic tissue issurgically accessed and the tissue therein examined visually and bypalpation for evidence of neoplastic tissue. The disclosed antibodiesradiolabeled with the disclosed radioisotopes are injected into thepatient at least 7 days, and preferably 7-21 days, prior to the surgeryso that during the surgery a probe may used to determine the presence ofradiolabeled antibody bound to neoplastic tissue.

[0115] The antibodies and radioisotopes of that reference, herebyincorporated by reference, are useful in the methods of the presentinvention wherein an injection of a radiolabeled antibody isadministered.

[0116] In the method of the present invention wherein intravascular,intraoperative, laparoscopic, or endoscopic procedures are used todetect and treat lesions and an injection is made of only one labeledprotein, any label disclosed herein as useful for labeling eitherfragments or whole antibodies may be used.

[0117] Suitable radioisotopes include, e.g., Cobalt-57, Iodine-131,Iodine-123, Iodine-124, Iodine-125, Iodine-126, Iodine-133, Bromine-77,Indium-111, Indium-113m, Copper-67, Gallium-67, Gallium-68,Ruthenium-95, Ruthenium-97, Ruthenium-103, Ruthenium-105, Mercury-197,Mercury-203, Rhodium-99m, Rhodium-101, Rhodium-105, Selenium-75,Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165,Thulium-167, Thulium-168, Rhenium-186, Rhenium-188, Technetium-99m,Fluorine-18, and Zirconium-97. Preferred are Technetium-99m, Iodine-123,Iodine-125, Iodine-131, Indium-111, and Gallium-67. Examples ofbeta-emitters are Phosphorus-32, Yttrium-90, Copper-67, Rhenium-186,Rhenium-188 and others.

[0118] The probe can be combined with surgical removal of detected tumoror lesions, cells, or tissue, and this is the conventional mode ofoperation in second-look surgery. Another possibility for such surgery,and especially for endoscopic and laparoscopic procedures, is to combinethe probe with a laser device that could use the signal generated by theprobe to indicate where laser irradiation should be directed toselectively destroy tumor tissue.

[0119] Suitable laser devices, combined with fiber optic transmission,are well known in the art. Representative examples thereof aredescribed, inter alia, in, e.g., Dixon, Lasers in Surgery, in “CurrentProblems in Surgery”, Pgs. 1-65 (Year Book Medical Pubs., Inc. 1984);Fleisher, Arch. Intern. Med., 144, 1225-1230 (1984); Wood et al., Am. J.Gastroent., 80, 715-718 (1985); and Hunter et al., Am. J. Surg., 148,736-741 (1984). Three types of lasers are currently in fairly widespreaduse for medical therapy, namely the argon, carbon dioxide andneodymium-YAG (yttrium aluminum garnet) lasers. As noted by Fleischer,Nd-YAG and argon lasers have been used with fiber optic waveguides,although it is likely that further advances in CO₂laser technology willpermit its use with fiber optics in the near future.

[0120] The foregoing references show that lasers have been used fortherapy, in conjunction with endoscopy, both in coagulative and inablative modes, including their use for tumor therapy. The use of lasersand endoscopy has advantages where surgery is contraindicated. Used inconjunction with the endoscopic procedure according to the presentinvention, greater precision and mitigation of damage to normal tissueis achieved.

[0121] The probe can also be used to provide radiation therapy such asbrachytherapy and external beam radiation to the site.

[0122] However, it normally will not be possible to optimize scanning inthe same way for intraoperative, intravascular or laparoscopic orendoscopic examination as it is for external imaging. Rather, in theevent that it is desired to optimize the timing of the procedure, ablood sample can be taken at periodic intervals after injection of theprotein and the level of activity of the label in the blood isdetermined, so that the efficacy of clearance and the level ofcirculating label can be observed. This will indicate the best time forthe surgical, intravascular, or laparoscopic or endoscopic procedure.For example, when the level of circulating label being detected isreduced by at least about 50%, preferably at least about 75%, morepreferably by at least about 85%, or even more, the interference ofbackground radiation with the short-range detection process will beminimized, and detection will be enhanced.

[0123] The invention provides a kit suitable for use in anintraoperative, intravascular, laparoscopic, or endoscopic procedure.

[0124] The kit may comprise a vial containing a sterile preparation forhuman use comprising an antibody fragment capable of being labeled withan isotopic or non-isotopic agent, and a vial containing a sterilepreparation for human use comprising an antibody capable of beinglabeled with a differing labeling agent.

[0125] The kit may also comprises a vial containing a sterile injectablepreparation for human use comprising a labeled antibody fragment and apharmaceutically acceptable carrier, and a vial containing a sterileinjectable preparation for human use comprising an antibody labeled witha differing labeling agent and a pharmaceutically acceptable carrier.

[0126] The labeled antibody fragment and labeled antibody are eachconveniently provided as an injectable preparation for mammalian use,preferably a sterile injectable preparation for human use, preferablycomprising: a sterile injectable solution containing an effective amountof the labeled antibody fragment or labeled antibody in apharmaceutically acceptable sterile injection vehicle, preferablyphosphate-buffered saline injection vehicle (PBS) at physiological pHand concentration. Other conventional pharmaceutically acceptablevehicles for parenteral administration may be utilized as required forthe site of parenteral administration.

[0127] A representative preparation to be parenterally administered inaccordance with this invention will normally contain about 0.01 to 20mg, preferably about 0.05 to 5 mg, of labeled antibody fragment orlabeled antibody, in a sterile solution which advantageously alsocontains, e.g., about 10 mg of human serum albumin (1% USP, Parke-Davis)per ml of 0.04M phosphate buffer (pH 7.4 Bioware) containing 0.9 sodiumchloride.

[0128] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.In the following examples, all temperatures are set forth uncorrected indegrees Celsius; unless otherwise indicated, all parts and percentagesare by weight.

[0129] The biopsy technique of the present invention involves exposingsuspicious areas using a scalpel, cutting a representative specimen fortissue analysis by means of histology. Alternatively, in anothersituation, a special biopsy instrument or a needle probe is used toobtain the tissue sample.

EXAMPLES Example 1 Intraoperative Tumor Detection

[0130] A female patient with a cecal carcinoma recurrence is injectedi.v. with Tc-99m-labeled murine monoclonal antibody NP-4 Fab′ fragmentagainst carcinoembryonic antigen (CEA) (15 mCi Tc-99m; 1 mg Fab′). Thepatient is scanned with a gamma camera, producing both planar andsingle-photon emission computed tomographic images at 3-5 hours later,and the cecal lesion is defined. Without delaying the planned surgery,the patient is brought to the operating room 8 hours after the antibodyadministration, and undergoes surgery for removal of the cecal tumor andany other sites of involvement in the region. This is achieved bypassing a sterile radiation probe over the viscera to identify sites oftumor spread. The probe has a cadmium telluride scintillation crystal,collimator, preamplifier and amplifier, similar to that reported byAitken et al., op. cit., but is collimated to select energy in the rangeof 100-160 keV. The counts registered are translated into a digitalrecording and an auditory signal at a significant increase overbackground counts. An increase in count ratio and signal of at least 60%relative to non-tumor areas is noted at the cecal tumor site, andextends for a distance of about 3 cm from the gross margins of thetumor. The surgeon removes the tumor and normal-appearing colonextending a distance of about 3.5 cm from the gross margins of the tumoron all sides, at which distance the ratio of counts was not more than25% higher than the normalized base ratio. Also, a mesenteric lymph nodegiving at least a 100% increased signal over background is alsoresected. The resected tissues are fixed in formalin and are found byhistopathology to contain carcinoma cells. The colon tumor borders arefound to be devoid of cancer invasion. Based upon follow-up of thepatient for 7 months, no evidence of local or distant cancer recurrencesis found, and the blood CEA titer, which is elevated prior to surgery,has remained within the normal range not exceeding 2.5 ng/ml.

Example 2 Endoscopic Tumor Detection

[0131] (A) A male patient with a suspected colonic polyp (having ahistory of recurrent polyps in the colon)is scheduled for a colonoscopybecause of a recent positive guaiac test for hemoglobin in his stool,and an elevated blood CEA titer of 12.5 ng/ml. A dose of CEA monoclonalantibody NP-4 F(ab′)₂ labeled with I-125 by the chloramine-T method (2mg F(ab′)₂with 1.0-mCi I-125) is injected i.v. and 24 hours later,without delaying the planned endoscopic procedure, the patient undergoesendoscopy, using a colonoscope equipped with a radiation detectorcapable of measuring the radiation emitted by I-125. The detectorcomprises a cadmium telluride scintillation crystal mounted on the tipof an optical fiber waveguide. The optical fiber is housed within ashielded tube which itself is inserted inside the colonoscope andextends to within about 4 mm of the open end thereof, the remaininglength of tubing serving as a collimator. The other end of the opticalfiber leads to a photomultiplier, a preamplifier and amplifier, andmeans to convert the resultant signal to a corresponding auditorysignal. The gastroenterologist examining the colon with the colonoscopefinds 2 small pedunculated polyps within a distance of 20 and 25 cm fromthe anal verge, measuring about 1.5 cm and 0.8 cm in diameter,respectively. The radiation detector shows an enhanced signal only overthe larger polyp, this signal appearing 1.8 times higher than backgroundactivity or the radioactivity over the other, smaller, polyp. Bothpolyps are removed by means of a loop inserted in the colonoscope,extracted through the scope, and processed for histopathology. It isfound that only the one having the high radioactivity signal containscancer cells; this polyp has an area of adenocarcinoma.

[0132] (B) In another case of endoscopy similar to the above, a patientreceives a monoclonal antibody to carcinoembryonic antigen (CEA) whichis conjugated to dihematoporphyrin ether, DHE (Photofrin II), by anadaptation of the carbodiimide-conjugation method of Mew et al., J.Immunol. 130:1473, 1983, at a dose of 1.5 mg DUE per kg body weight i.v.The fiberoptic colonoscope used to detect the fluorescence of the agenttargeted to a malignant polyp is similar to the fiberoptic bronchoscopedescribed by Profio et al., Adv. Exp. Med. Biol. 193:43, 1985, using aviolet krypton ion laser emitting at 410 nm. The fluorescence intensityis converted to an audio signal, whose pitch is related to the signal'sintensity by analyzing the fluorescence in a photomultiplier tube. Inthis case, the malignant polyp at a distance of 15 cm from the analverge has a ratio of fluorescence to background of 6:1. The polyp of 0.5cm in diameter is removed via the colonoscope, fixed in formalin, andprocessed for histopathology. An adenocarcinoma in the stalk of thepolyp is found to be present.

Example 3 Intraoverative Tumor Therapy

[0133] A woman with ovarian cancer having extensive abdominal spread isinjected prior to surgery with a biotinylated preparation of RS7-3G11monoclonal antibody (3 mg) i.v. Three days' later a chase dose of avidin(10 mg) is given i.v. in 2 divided doses 60 minutes apart. Twenty-fourhours later, a 1.5 mg dose of biotinylated RS7-3G11 conjugated withDTPA-indium (stable) is injected i.v. The next day, the patientundergoes a resection of all visible and palpable tumors in herabdominal cavity, followed by intraoperative irradiation of the exposedcavity with monochromatic X-rays of 40 keV to destroy micrometastaticcancer spread. At 6 and 9 months later, no evidence of disease ispresent, and the patient's blood CA-125 titer is within the normalrange, as contrasted to its marked elevation prior to treatment

Example 4 Intravascular Detection and Therapy of a Thrombosed MyocardialArtery

[0134] A male presents with disabling angina pectoris and suspectedcoronary artery disease. It is decided to examine the coronary arteriesand a catheter is inserted by the percutaneous femoral technique. Thecatheter had a fiberoptic system similar to the ones used in colonoscopyabove, but miniaturized with transmission of fluorescence and laserlight without the need to visualize the tissues, only to measurefluorescence emitted. A similar DHE preparation as in Example 2(B),conjugated to monoclonal antibody EPB-1 (LL1), which targetsatherosclerotic plaques, is injected at a dose of 1.0 mg per kg bodyweight i.v. Using an excimer-dye laser which emits 405 nm laser beamsfor diagnosis, the catheter finds an increased signal of fluorescence(translated into an auditory signal whose pitch reflects fluorescenceintensity, using a photomultiplier) in the patient's left anteriordescending artery. Thereafter, the laser emits a 630 nm laser beamthrough the optical fiber in the catheter for dissolving theatherosclerotic plaque, keeping the fiber input energy below 4 mJ/pulse.Follow-up reveals that the occluded vessel improved its blood flow by atleast 70% without any adverse effects to the patient. The patient'ssymptoms and cardiac status improves in the ensuing 8 weeks.

[0135] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

[0136] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method for close-range lesion detection, duringan operative, intravascular, laparoscopic, or endoscopic procedure,wherein the method comprises: (a) injecting a patient subject to such aprocedure parenterally with an effective amount of a labeled divalentsingle chain antibody fragment or subfragment with a molecular weight of85,000 daltons or less, which specifically binds an antigen produced byor associated with a lesion; (b) conducting the procedure within 48hours of the injection; (c) scanning the accessed interior of thepatient at close range with a detection means for detecting the presenceof the labeled antibody fragment or subfragment; and (d) locating thesites of accretion of the labeled antibody fragment or subfragment bydetecting elevated levels of the labeled antibody fragment orsubfragment at such sites with the detection means.
 2. The method ofclaim 1, wherein said the procedure is conducted within 24 hours of theinjection.
 3. The method of claim 1, wherein the procedure is conductedwithin 12 hours of the injection.
 4. The method of claim 1, wherein theprocedure is conducted within 6 hours of the injection.
 5. The method ofclaim 1, wherein the lesion is selected from the group consisting of acancer, an infectious lesion, an inflammatory lesion, a non-tumorous ornoninfectious inflammation, a clot, hyperplasia and atheroscleroticplaque.
 6. The method of claim 1, wherein the fragment or subfragment ismonospecific.
 7. The method of claim 1, wherein the fragment orsubfragment is bispecfic.
 8. The method of claim 1, wherein the fragmentor subfragment has a molecular weight of 65,000 daltons or less.
 9. Themethod of claim 1, wherein the fragment or subfragment has a molecularweight of 55,000 daltons or less.
 10. The method of claim 1, wherein thefragment or subfragment has a molecular weight of 50,000 daltons orless.
 11. The method of claim 1, wherein the label for the fragment orsubfragment is a radioisotope that emits at an energy of 20-1,000 kev.12. The method of claim 11, wherein the radioisotope is selected fromthe group consisting of technetium-99m, iodine-125, iodine-131,iodine-123, indium-111, and gallium-67.
 13. The method of claim 1,wherein the label of said labeled antibody fragment or subfragment is anon-isotopic agent.
 14. The method of claim 13, wherein the non-isotopicagent is a photoactive agent.
 15. The method of claim 14, wherein thephotoactive agent is a fluorescent agent.
 16. The method of claim 1,wherein the method further comprises treating detected sites during theprocedure.
 17. The method of claim 1, wherein the procedure is anoperative procedure.
 18. The method of claim 1, wherein the procedure isan intravascular procedure.
 19. The method of claim 1, wherein theprocedure is an endoscopic procedure.
 20. The method of claim 1, whereinsaid procedure is a laparoscopic procedure.
 21. The method of claim 1,wherein said procedure is a biopsy, and the biopsy implement is guidedto lesions at sites of elevated label accretion.
 22. The method of claim1, further comprising treating the lesion during the procedure bybrachytherapy, external beam radiation, laser therapy or surgicalremoval.
 23. The method of claim 1, wherein the location of sites ofaccretion is performed without the use of a clearing agent, contrastagent or subtraction agent.
 24. A method of detection of lesions duringan endoscopic, laparoscopic, intravascular catheter, or surgicalprocedure, wherein the method comprises: injecting a patient to undergosuch a procedure with a divalent single chain antibody fragment orsubfragment with a molecular weight of 85,000 Daltons or less, labeledwith a fluorescent agent or dye, wherein the labeled antibody fragmentor subfragment accretes at the lesion; permitting the labeled antibodyfragment or subfragment to accrete; conducting the procedure within 48hours of the injection; and detecting the label with a light sourcesupplied via an endoscope, laparoscope, or intravascular catheter orduring the surgical procedure.
 25. The method of claim 24, furthercomprising the step of removing lesions at sites of elevated labelaccretion with a laser or surgically.
 26. The method of claim 25,further comprising the step of treating lesions at sites of elevatedlabel accretion with ionizing radiation.
 27. The method of claim 26,wherein the procedure is selected from the group consisting of anendoscope, laparoscope, and intravascular catheter procedures, furthercomprising the step of administering brachytherapy via the endoscope orcatheter to lesions at sites of elevated label accretion.
 28. The methodof claim 24, wherein the lesion is selected from the group consisting ofa cancer, an infectious lesion, an inflammatory lesion, a non-tumorousor noninfectious inflammation lesion, a clot, hyperplasia andatherosclerotic plaque.
 29. The method of claim 24, wherein the fragmentor subfragment is monospecific.
 30. The method of claim 24, wherein thefragment or subfragment is bispecfic.
 31. The method of claim 24,wherein the fragment or subfragment has a molecular weight of 65,000daltons or less.
 32. The method of claim 24, wherein the fragment orsubfragment has a molecular weight of 55,000 daltons or less.
 33. Themethod of claim 24, wherein the fragment or subfragment has a molecularweight of 50,000 daltons or less.
 34. The method of claim 24, whereinsaid procedure is conducted within 24 hours of the injection of saidlabeled antibody fragment or subfragment.
 35. The method of claim 24,wherein said procedure is a laparoscopic procedure.
 36. The method ofclaim 24, wherein the label is detected without the use of a clearingagent, contrast agent or subtraction agent.
 37. A method of detectionand treatment of lesions during an endoscopic, laparoscopic, orintravascular catheter procedure, wherein the method comprises: (a)injecting a patient to undergo such a procedure with a divalent singlechain antibody fragment or subfragment with a molecular weight of 85,000Daltons or less, labeled with an agent capable of detection, whichlabeled antibody fragment or subfragment preferentially accretes at thelesion; (b) permitting the labeled antibody fragment or subfragment toaccrete at the lesion; (c) conducting the procedure within 48 hours ofthe injection; (d) detecting the agent with a detection means suppliedvia the endoscope, laparoscope, or intravascular catheter; and (e)treating the lesion by brachytherapy administered through the endoscopeor intravascular catheter.
 38. The method of claim 37, wherein thelesion is selected from the group consisting of a cancer, an infectiouslesion, an inflammatory lesion, a non-tumorous or noninfectiousinflammation lesion, a clot, hyperplasia and atherosclerotic plaque. 39.The method of claim 37, wherein the fragment or subfragment ismonospecific.
 40. The method of claim 37, wherein the fragment orsubfragment is bispecfic.
 41. The method of claim 37, wherein thefragment or subfragment has a molecular weight of 65,000 daltons orless.
 42. The method of claim 37, wherein the fragment or subfragmenthas a molecular weight of 55,000 daltons or less.
 43. The method ofclaim 37, wherein the fragment or subfragment has a molecular weight of50,000 daltons or less.
 44. The method of claim 37, wherein saidprocedure is conducted within 24 hours of the injection of said labeledantibody fragment or subfragment.
 45. The method of claim 37, whereinsaid procedure is a laparoscopic procedure.
 46. The method of claim 37,wherein the label is detected without the use of a clearing agent,contrast agent or subtraction agent.
 47. A method of treatment oflesions during a laparoscopic or intravascular catheter procedure,wherein the method comprises: injecting a patient to undergo such aprocedure with a divalent single chain antibody fragment or subfragmentwith a molecular weight of 85,000 Daltons or less, labeled with aphotoactive agent, wherein the antibody fragment or subfragmentpreferentially accretes at targeted lesions; permitting the labeledantibody fragment or subfragment to accrete; conducting the procedurewithin 48 hours of the injection; and activating the photoactive agentwith a light source supplied via the laparoscope or intravascularcatheter, thereby treating said lesions.
 48. The method of claim 47,wherein the lesion is selected from the group consisting of a cancer, aninfectious lesion, an inflammatory lesion, a non-tumorous ornoninfectious inflammation lesion, a clot, hyperplasia andatherosclerotic plaque.
 49. The method of claim 47, wherein the fragmentor subfragment is monospecific.
 50. The method of claim 47, wherein thefragment or subfragment is bispecfic.
 51. The method of claim 47,wherein the fragment or subfragment has a molecular weight of 65,000daltons or less.
 52. The method of claim 47, wherein the fragment orsubfragment has a molecular weight of 55,000 daltons or less.
 53. Themethod of claim 47, wherein the fragment or subfragment has a molecularweight of 50,000 daltons or less.
 54. The method of claim 47, whereinsaid procedure is conducted within 24 hours of the injection of saidlabeled antibody fragment or subfragment.
 55. The method of claim 47,wherein said procedure is a laparoscopic procedure.
 56. A method oftreatment of lesions, wherein the method comprises: (a) injecting apatient with composition comprising a divalent single chain antibodyfragment or subfragment with a molecular weight of 85,000 Daltons orless, conjugated to an agent capable of being activated to emit Augerelectrons or other ionizing radiation, and, optionally, to an agentcapable of detection, wherein the antibody conjugate accretespreferentially at the targeted lesion; and (b) activating said agentcapable of being activated, thereby treating said lesions, and,optionally, detecting the optional agent capable of detection.
 57. Themethod of claim 56, wherein the activation and optional detection isduring an endoscopic, intravascular, catheter or surgical procedure. 58.The method of claim 56, wherein said procedure is a laparoscopicprocedure.
 59. The method of claim 56, wherein the activatable agent isa stable element capable of being activated to emit ionizing radiation.60. The method of claim 56, wherein the activatable agent is a stableelement capable of being activated to emit Auger electrons.
 61. Themethod of claim 56, wherein the activatable agent is a halogenatedcompound.
 62. The method of claim 61, wherein the agent is a halogenatedpyrimidine.
 63. The method of claim 59, wherein the stable element isiodine or indium.
 64. The method of claim 56, wherein the activatingenergy is monochromatic X-rays.
 65. The method of claim 64, wherein themonochromatic X-rays emit at an energy of 20-70 keV.
 66. The method ofclaim 65, wherein the monochromatic X-rays emit at an energy of 30-40keV.
 67. The method of claim 56, wherein the lesion is selected from thegroup consisting of a cancer, an infectious lesion, an inflammatorylesion, a non-tumorous or noninfectious inflammation lesion, a clot,hyperplasia and atherosclerotic plaque.
 68. The method of claim 56,wherein the fragment or subfragment is monospecific.
 69. The method ofclaim 56, wherein the fragment or subfragment is bispecfic.
 70. Themethod of claim 56, wherein the fragment or subfragment has a molecularweight of 65,000 daltons or less.
 71. The method of claim 56, whereinthe fragment or subfragment has a molecular weight of 55,000 daltons orless.
 72. The method of claim 56, wherein the fragment or subfragmenthas a molecular weight of 50,000 daltons or less.
 73. A method ofobtaining biopsy samples, wherein the method comprises: (a) injecting apatient subject to such a procedure parenterally with an effectiveamount of a labeled antibody fragment or subfragment, which specificallybinds an antigen produced by or associated with a lesion; (b) scanningthe accessed interior of the patient at close range with a detectionmeans for detecting the presence of the labeled antibody fragment orsubfragment; (c) locating the sites of accretion of the labeled antibodyfragment or subfragment by detecting elevated levels of the labeledantibody fragment or subfragment at such sites with the detection means;and (d) inserting a biopsy implement into one or more sites of elevatedaccretion to obtain a biopsy sample, wherein said locating and saidbiopsy are conducted within 48 hours of the injection.
 74. The method ofclaim 73, wherein the procedure is conducted within 24 hours of theinjection.
 75. The method of claim 73, wherein the procedure isconducted within 12 hours of the injection.
 76. The method of claim 73,wherein the procedure is conducted within 6 hours of the injection. 77.The method of claim 73, wherein the lesion is selected from the groupconsisting of a cancer, an infectious lesion, an inflammatory lesion, anon-tumorous or noninfectious inflammation, a clot, hyperplasia andatherosclerotic plaque.
 78. The method of claim 73, wherein the fragmentor subfragment is a monoclonal antibody fragment or sub fragment. 79.The method of claim 73, wherein the fragment or subfragment ismonovalent and selected from the group consisting of an Fv, a singlechain antibody, Fab and Fab′.
 80. The method of claim 73, wherein thefragment or subfragment is a divalent single chain antibody fragment orsubfragment with a molecular weight of 85,000 daltons or less.
 81. Themethod of claim 73, wherein the fragment or subfragment is bispecfic.82. The method of claim 73, wherein the fragment or subfragment has amolecular weight of 65,000 daltons or less.
 83. The method of claim 82,wherein the fragment or subfragment has a molecular weight of 55,000daltons or less.
 84. The method of claim 82, wherein the fragment orsubfragment has a molecular weight of 50,000 daltons or less.
 85. Themethod of claim 73, wherein the label for the fragment or subfragment isa radioisotope.
 86. The method of claim 85, wherein the radioisotope isselected from the group consisting of technetium-99m, iodine-125,iodine-131, iodine-123, indium-111 and gallium-67.
 87. The method ofclaim 73, wherein the label of said labeled antibody fragment orsubfragment is a non-isotopic agent.
 88. The method of claim 87, whereinthe non-isotopic agent is a photoactive a gent.
 89. The method of claim88, wherein the photoactive agent is a fluorescent agent.
 90. The methodof claim 73, wherein the location of sites of accretion is performedwithout the use of a clearing agent, contrast agent or subtractionagent.
 91. A method of detection of lesions during an endoscopic,laparoscopic, intravascular catheter, or surgical procedure, wherein themethod comprises: (a) injecting a patient who is to undergo such aprocedure with a bispecific antibody F(ab)₂ or F(ab′)₂ fragment, whereinthe bispecific antibody fragment has a first antibody binding site whichspecifically binds to an antigen produced or associated with a lesion,and has a second antibody binding site which specifically binds to ahapten, and permitting the antibody fragment to accrete at target sites;(b) optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (c)detecting the presence of the hapten by detecting elevated levels ofaccreted label at the target sites with detection means, within 48 hoursof the first injection, and conducting said procedure.
 92. The method ofclaim 91, wherein said antigen produced or associated with a lesion is atumor- or pathogen-associated antigen.
 93. The method of claim 91,further comprising the step of removing lesions at sites of elevatedlabel accretion with a laser or surgically.
 94. The method of claim 91,further comprising the step of treating lesions at sites of elevatedlabel accretion with ionizing radiation.
 95. The method of claim 91,wherein the procedure is selected from the group consisting of anendoscope, laparoscope, and intravascular catheter procedures, furthercomprising the step of administering brachytherapy via the endoscope orcatheter to lesions at sites of elevated label accretion.
 96. The methodof claim 91, wherein the lesion is selected from the group consisting ofa cancer, an infectious lesion, an inflammatory lesion, a non-tumorousor noninfectious inflammation lesion, a clot, hyperplasia andatherosclerotic plaque.
 97. The method of claim 91, wherein saidprocedure is a laparoscopic procedure.
 98. The method of claim 91,wherein said hapten is labeled with a diagnostic radioisotope, a MRIimage enhancing agent or a fluorescent label.